As the technology of genetic engineering has advanced and the knowledge of molecular biology, the basis for that technology, has been accumulated rapidly, it has become possible to manipulate genes artificially and yet to introduce them into animals [Gordon, J. W. et al., Proc. Natl. Acad. Sci. U.S.A. 77:7380-7384 (1980)]. For example, methods for artificially adding to organisms genetic characters which are foreign to them or methods for inhibiting the expression of endogenous genetic characters in organisms have been developed. By utilizing such methods, animals into which various genetic characters are introduced have been created and reported as transgenic animals or knockout animals.
These transgenic animals are important from the view point that they make it possible to study the functions of various genes isolated and cloned by genetic engineering technologies at the individual level. So far, gene functions have been studied using ex vivo cultured cells, such as cell lines or primary culture cells, and findings obtainable from such study have been rather limited. In particular, experiments and researches have been vigorously made in which such transgenic animals are used for analyzing in vivo physiological functions of cloned genes or used as model animals for genetic diseases.
Embryonic stem cells (hereinafter, referred to as “ES cells”) are a cell strain which is established from the internal cell mass present inside of a blastocyst (early embryo after fertilization) and can be grown/cultured while retaining the undifferentiated state. These cells have pluripotency (totipotence) i.e. the ability to differentiate into any cell in the body. When injected into a normal early embryo, they are capable of participating in the formation of the embryo to generate a chimeric animal [Evans, M. J. and Kaufman M. H., Nature 292:154 (1981)].
Creation of various gene-mutated animals has been attempted utilizing this nature of ES cells. The history began with the establishment of ES cells by Evans and Kaufman in 1981, and earnest researches started with the creation of ES chimeric mice by Bradley et al. [Nature 309:255 (1984)]. Further, researches in this field have developed rapidly, e.g. homologous recombination of ES cells by Thomas and Capecchi [Cell 51:503 (1987)]; success in germ line transmission of ES cell characters by three research groups including Koller et al.; and creation of gene-deficient mice.
ES cell lines established so far include EK cells of Evans and Kaufman (supra); ES-D3 cells of Doetschman [J. Embryol. Exp. Molph. 87:27 (1981)]; CCE cells of Robertson [Nature 323:445 (1986)]; and BL/6III cells of Ladermann and Burki [Exp. Cell Res. 197:254]. Most of them are established from 129 strain mice.
As gene expression-deficient animals created with these ES cells, the following animals have been reported: (I) HPRT gene deficient mice created with spontaneous mutant ES cells by Hooper et al. [Nature 326:292 (1987)] and Knehn et al. [Nature 326:295 (1987)]; (2) p53 deficient mice which lack p53, one of tumor suppressor genes, by Donehower et al. [Nature 356:215 (1992)]; (3) β 2 microglobulin gene mutant mice by Zijlstra et al. [Nature 344:742 (1990)]; (4) RAG-2 (V(D)J recombination activation gene) mutant mice by Sinkai et al. [Cell 68:855 (1992)] which is one of disease model mice; (5) MHC class II mutant mice by Glimcher et al. [Science 253:1417 (1991)] and Cosgrove et al. [Cell 66:1051 (1991)]; (6) as one of development/growth related disease model mice, int-1 gene deficient mice by MacMahon et al. [Cell 62:1073 (1990)]; and (7) src deficient mice exhibiting osteopetrosis-like symptoms by Soriano et al. [Cell 64:693 (1991)].
On the other hand, a number of molecules, such as apoproteins [Srivastava R. & A. Srivastava, N., Mol. Cell Biochem. 209:131-144 (2999)], enzymes, receptors [Hiltumen, T. P. & Yla-Herttuala, S., Atherosclerosis 137:S81-88 (1988)] and lipid transfer proteins [Yamashita, S. et al., Atherosclerosis 152:271-285 (2000); Oram, J. F. & Vaughan, A. M., Curr. Opin. Lipidol., 11:253-260 (2000)], are involved in the metabolism of lipoproteins. Differences among individuals resulted from different genetic types of these proteins or brought by different diet are closely related to arteriosclerosis. High-density lipoprotein (HDL)-cholesterol, which is one of such proteins, is recognized by a number of epidemiological researches as an independent negative risk factor for coronary artery diseases [Barter, P. J. & Rye, K A., Atherosclerosis 121:1-12 (1996)]. HDL plays an important role in the so-called cholesterol reverse-transfer system which extracts excessive cholesterol from peripheral tissues and transfers the cholesterol to the liver [Tall, A. et al., Arterioscler. Thromb. Vasc. Biol., 20:1185-1188 (2000); Santamarina-Fojo, S. et al., Curr. Opin. Lipidol., 11:267-75 (2000)]. Lecithin:cholesterol acyltransferase (LCAT), which is the rate-determining enzyme in the system, is produced mainly in the liver [Warden, C. et al., J. Biol. Chem. 264:21573-21581 (1989)] and is present in HDL particles in blood [Kuivenhoven, J. et al., J. Lipid Res. 38:191-205 (1997)]. The free cholesterol extracted from peripheral cells by pre β HDL is esterified by LCAT, and thus the flow of cholesterol into cells is inhibited. As a result, LCAT exhibits cholesterol extraction-promoting effect, and it is believed that LCAT functions in an anti-arteriosclerotic manner [Czarnecka, H. & Yokoyama, S., Biochemistry 34:4385-92 (1995)]. Recently, an enzyme LLPL that has 47% homology to human LCAT at the amino acid level has been found by subtraction PCR using a cDNA library of human macrophage-like cells, and the full-length amino acid sequence thereof has been determined by cDNA analysis [Taniyama et al., Biochem. Biophys. Res. Commun. 257:50-56 (1999)].
Large quantities of macrophages transformed into foam cells are accumulated in human arteriosclerosis lesions. Besides, it is known that LLPL is secreted in human macrophage-like cells and that ApoE (apolipoprotein E) gene is one of the genes associated with arteriosclerosis. The results of in situ hybridization using arteriosclerosis lesions of an arteriosclerosis model mouse (ApoE deficient mouse) revealed that mouse LLPL gene is expressed in those lesions. Thus, it is considered that LLPL gene is closely related to the progress of arteriosclerosis.
Although human LLPL does not have the LCAT activity to esterify free cholesterol, it exhibits the lysophospholipase activity to degrade free cholesterol into free fatty acids and glycerophosphorylcholine in vitro using lysophosphatidylcholine as a substrate [Taniyama et al., Biochem. Biophys. Res. Commun. 257:50-56 (1999)]. Further, it has been also confirmed that human LLPL is present in human serum. The presence of unidentified substrate in serum is suggested.
It is known that LLPL is secreted in macrophage-like cells in human and in peritoneal macrophages in mouse; and that human LLPL and mouse LLPL are 88% homologous in the amino acid sequence and their lipase motifs are the same AHSMG sequence (Japanese Unexamined Patent Publication No. 11-269199). In both human and mouse, LLPL gene is expressed specifically in peripheral tissues. Thus, human LLPL and mouse LLPL have a number of similarities. Further, since expression of LLPL is confirmed in arteriosclerosis lesions of ApoE-deficient mice, it is believed that LLPL has effects upon arteriosclerosis lesions and plasma lipid profile.
However, these effects have not been elucidated sufficiently and there are many issues of which should be clarified with regard to in vivo functions of LLPL and the mechanism thereof. Animal models deficient in expression of LLPL gene which produce no or little LLPL are indispensable for elucidating the in vivo effects of LLPL and desired eagerly. However, no such animal models have been created to date.
Therefore, if non-human animal ES cells where their LLPL gene is inactivated have been successfully created, it is possible to create non-human animals deficient in expression of LLPL gene. Since the resultant non-human animals deficient in expression of LLPL gene lack various biological activities inducible by LLPL, they can be models for such diseases resulting from inactivation of the biological activities of LLPL. With such animal models, it becomes possible to elucidate the causes of such diseases and to examine therapeutic methods for such diseases.